Type 1 diabetes (T1DM) alters carbohydrate, amino acid, and fatty acid metabolism contributing to nephropathy, retinopathy and peripheral neuropathy, three of the most debilitating complications of diabetes. The prevailing view suggests that systemic hyperglycemia drives complications by similar biochemical mechanisms in all tissues. However, this has not been specifically tested, especially in vivo. In these studies, we propose that the normal cellular nutrient utilization in cells of complication-prone tissues is distinct and therefore reaction to insulin deficiency, hyperglycemia and other changes of T1DM will also be distinct. Indeed, our preliminary data demonstrate tissue-specific changes in cellular metabolite levels that are not driven by mass-action alone but appear to be due to selective metabolic reprogramming of the target tissues. In contrast to increased levels in kidney, glycolysis and tricarboxylic acid cycle (TCA) cycle intermediates are decreased in diabetic mouse nerve and retina despite ambient hyperglycemia. Importantly, the increased mouse kidney and human urinary levels of TCA metabolites predict progression of diabetic nephropathy, suggesting that metabolic reprogramming may play a pathogenic role in the progression of complications. These findings lead to our hypothesis that diabetic complications arise from tissue-specific metabolic reprogramming resulting in alterations in fuel utilization which lead to dysfunction of the tissue. To test this hypothesis, we will use sensitive and specific mass spectrometer based metabolomic analysis in models of diabetes to define changes in metabolite levels and flux in three complications-prone tissues, retina, kidney and peripheral nerves. We will extend these studies to humans with T1DM to understand intrinsic differences from non-diabetics in metabolite levels and flux in the kidney. We will define the changes in mRNA and protein expression and post-translational protein modification to determine the basis for altered metabolite levels. Finally, we will utilize appropriately engineered mouse animals to directly test the refined hypotheses arising from these studies. Our specific aims are to: Aim 1: Identify the alterations in steady state abnormalities of intermediary metabolism in kidney, nerve and retina in the best murine models of diabetic complications using state-of-the-art metabolomic approaches Aim 2: Determine metabolite flux in all 3 tissues from the murine models and identify the key regulatory reactions that contribute to the metabolite abnormalities. Aim 3: Assess steady state and dynamic metabolite changes in humans with type 1 diabetes with and without microvascular complications. Aim 4: Define regulatory mechanisms of altered cellular metabolism in complication-prone tissues and test their effect in murine models. PUBLIC HEALTH RELEVANCE: This proposal will test the hypothesis that diabetic complications arise from specific changes in cellular substrate metabolism which can be defined using modern molecular phenotyping techniques in both animal models and in humans and that interventions to modulate specific metabolic pathways may mitigate or prevent development and progression of these complications. Our study is designed to gain a better understanding of the changes in metabolite levels and flux in complications-prone tissues in animal models and patients with type 1 diabetes mellitus and to determine how the metabolite changes reflect altered levels or activities of specific proteins and lipids which contribute to 'microvascular'complications. We will utilize in vivo and in vitro analysis of mouse models and human patients to achieve these goals.